Okadaic Acid In Mussels Research Articles

Eavesdropping on plankton—can zooplankton monitoring improve forecasting of biotoxins from harmful algae blooms?

AbstractHarmful algae bloom (HAB) forecasting has developed rapidly over recent decades, but predicting harmful levels of marine biotoxins in shellfish is still a challenge. New discoveries suggest that predator‐prey interactions may be an important driver in the formation of HABs. Key species of harmful algae respond to copepod infochemicals with increased toxin production. In addition, copepods feed selectively on less defended prey, which may further promote harmful taxa. Here we explore if eavesdropping on predator‐prey dynamics by monitoring zooplankton can improve HAB forecasting. We first examine an 8‐yr time series including copepod biomass, harmful algae cells (Dinophysis spp.), and diarrhetic shellfish toxins in blue mussels (Mytilus edulis) using generalized additive models. Models including copepod biomass more accurately predicted okadaic acid in mussels than phytoplankton alone. We then apply this connection more narrowly by analyzing the specific copepod exudates known to induce toxin production, copepodamides, from the mussels sampled in biotoxin monitoring. Adding copepodamide data from shellfish extracts increased model performance compared to copepod biomass. Results suggest that including grazing effects through copepodamide measurements may provide a cost‐efficient way to improve accuracy and lead time for predicting the accumulation of microalgal toxins in shellfish.

A novel microfluidic system for the sensitive and cost-effective detection of okadaic acid in mussels.

Okadaic acid (OA) is produced by marine dinoflagellates and it can be easily accumulated in shellfish, causing intoxications when consumed by humans. Consequently, there is a need for sensitive, reliable and cost-effective methods to detect OA in real samples. In this work, we developed a novel and affordable microfluidic system to detect OA based on the protein phosphatase 1 inhibition colorimetric assay. This enzyme was immobilized in a microfluidic chamber by physisorption in an alumina sol-gel. The results show good enzyme stability over time when maintained at 4 °C. The developed system was sensitive for OA standard solutions, presenting a limit of detection (LOD) of 11.6 nM over a large linear range (43.4 to 3095.8 nM). Our method revealed an LOD as low as 0.2 μg kg-1 and a linear range between 1.47 and 506 μg kg-1 for extracted mussel matrix, detecting OA concentrations in contaminated mussels much lower than the regulated limit (160 μg kg-1). The enzyme stability and reusability along with the simplicity and low cost associated with microfluidics systems make this method very interesting from a commercial point of view.

SPR immunosensor for the detection of okadaic acid in mussels using magnetic particles as antibody carriers

Protein G-coated magnetic particles (MPs) were used as immobilisation supports for an antibody against okadaic acid (MAbOA) and carriers into a surface plasmon resonance (SPR) device for the development of a direct competitive immunosensor for okadaic acid (OA). SPR analysis of MAbOA–MP conjugates demonstrated that conjugations were successful with complete immobilisation of all the antibody biomolecules onto the MPs. Moreover, MAbOA–MP conjugates provided up to 11-fold higher SPR signals, compared to free MAbOA. The use of conjugates in the direct competition assay provided a 3-fold lower LODμg/L (2.6μg of OA/L, equivalent to 12μg of OA/kg mussel meat). The presence of mussel matrix did not interfere in the OA quantification as seen in the calibration curves. Mussel samples, obtained from Ebro Delta's bays (NW Mediterranean) during a diarrheic shellfish poisoning (DSP) event and in the presence of Dinophysis sacculus, an OA producer, in the shellfish production area, were analysed with the MP-based SPR immunosensor. The OA contents correlated with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (y=0.984x−5.273, R2=0.789, p<0.001) and by mouse bioassay (MBA).

Evaluating the performance of sampling plans to detect okadaic acid in mussels in production areas

INTRODUCTION: Bivalve molluscan shellfish feed on phytoplankton (micro-algae) which might potentially contain biotoxins like okadaic acid. In order to ensure food safety, the level of okadaic acid equivalents in live bivalve molluscs must not exceed 160 μg per kg (Regulation 853/2004/EC). Therefore, sampling plans must be set by official authorities in order to monitor production areas to check the presence of okadaic acid in shellfish, knowing that mussel may be used as an indicator species (Regulation 854/2004/EC). There is a high interindividual variability between mussels, even for mussels taken at the same sampling point and at the same time (Edebo et al., 1988 ; Duinker et al., 2007). However there has never been a probabilistic evaluation of the impact that the sample size and the number of samples taken, have on the analysis results and the corresponding decision for the shellfish lot. Nathalie Wesolek (1), Francois-Gilles Carpentier (1), Dominique Parent-Massin (1), Alain-Claude Roudot (1) (1) Laboratoire de Toxicologie Alimentaire et Cellulaire, Universite de Bretagne Occidentale, UBO, 29200 BREST (email : nathalie.wesolek@univ-brest.fr)

First Evidence of Okadaic Acid in Mytilus galloprovincialis Mussels, Collected in a Mediterranean Lagoon, Tunisia

Evidence of the presence of okadaic acid (OA) and its monthly fluctuations are reported for the first time in Mytilus galloprovincialis samples collected from June 2005 to May 2006 in the Bizerte Lagoon. All of the samples with the exception of those taken in August 2005 were found to be contaminated. The level of OA in mussels exceeded the regulatory limit of DSP toxins (16 microg OA/100 g mussel meat) within the European Union (2002) in only two cases: in January and February, 2006 with 31.85 +/- 3.06, and 18.86 +/- 1.88 microg OA/100 g mussel meat respectively. During the investigative period, potentially toxic dinoflagellates were observed. The results reported here, although based on limited sampling, demonstrate for the first time the existence of a risk to public health from diarrheic shellfish poisoning in cultivated bivalve mollusc in Tunisia.

Detection of the marine toxin okadaic acid in mussels during a diarrhetic shellfish poisoning (DSP) episode in Thermaikos Gulf, Greece, using biological, chemical and immunological methods

An approach involving chemical and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos Gulf, Greece, during a 4-month DSP episode that occurred in 2002. Samples were analyzed using the mouse bioassay, high performance liquid chromatography (HPLC) with fluorimetric detection and an enzyme-linked immunosorbent assay (ELISA). Okadaic acid was quantifiable at three different sites of Thermaikos Gulf reaching a maximum concentration of 36 μg/g hepatopancreas. High correlation was revealed between results derived from HPLC and ELISA ( R 2 = 0.998), while 91% consistency between HPLC and the mouse bioassay results was observed.

Analysis of the importance of lipid breakdown for elimination of okadaic acid (diarrhetic shellfish toxin) in mussels, Mytilus edulis: results from a field study and a laboratory experiment

Okadaic acid (OA) is a lipophilic phycotoxin, which accumulates in the digestive organs of mussels and may cause diarrhetic shellfish poisoning (DSP) in humans. Depuration of toxic mussels is a potential option for the shellfish industry to increase the availability of marketable mussels. To develop cost-effective depuration methods for DSP toxins, knowledge about the environmental conditions and physiological processes regulating the rate of depuration is essential. In this paper, the importance of lipid breakdown for elimination of OA in mussels was investigated by performing a field study and a manipulative laboratory experiment. First, total lipid content and concurrent concentration of OA in the digestive glands of farmed blue mussels, Mytilus edulis, was analysed on a monthly basis from January to June 2000. A significant positive correlation between levels of OA and lipid content was observed between January and March, when lipid levels were showing a decreasing trend. This supported a previously proposed model that breakdown of lipid stores may affect the release and elimination of this lipophilic toxin. To test this causal model, a laboratory experiment was performed. Mussels containing OA were exposed to experimental treatments (increased seawater temperature and/or food limitation) for 24 days in order to increase the energy requirements and need to use lipids as an energy source. It was predicted that mussels exposed to these treatments would have a faster elimination rate of OA compared to feeding mussels kept in ambient seawater temperature. The results showed that lipid content was significantly reduced in mussels exposed to an increased water temperature (24 °C) compared to ambient temperature (18 °C). The amount of lipids was not affected by food limitation. Although lipid content was reduced in 24 °C, the rate of depuration of OA was not faster for mussels in this treatment and no correlation was detected between lipid content and OA. Depuration rates were very similar for all treatments and followed an exponential decrease relationship ( t 1/2=8 days). Thus, the proposed model that lipid breakdown affects the mechanism of elimination of OA was not supported. Nevertheless, the observed rates of depuration provide useful information and a potential predictive tool for large-scale depuration methods of mussels. The difficulties to influence the rate of depuration of this toxin by changing the environmental conditions suggest that processes, insensitive to short-term manipulation of the external environment, regulate depuration of OA.

Mussel blood cells, resistant to the cytotoxic effects of okadaic acid, do not express cell membrane p-glycoprotein activity (multixenobiotic resistance)

Okadaic acid (OA) is a dinoflagellate toxin, accumulating in shellfish and causing diarrhetic shellfish poisoning (DSP) in humans. OA is a highly cytotoxic agent in most cell lines because of its inhibiting properties of protein phosphatases. So far, the cytotoxicity of OA in mussels, the main vectors of DSP, has not been investigated. In this paper, the viability of mussel ( Mytilus edulis) blood cells incubated in 10 nM–1 μM OA was studied. After 72 h of exposure, viability was reduced to 54% in 1 μM OA compared with 88% in control cells. This yielded a LC 50 of >1 μM for OA, which is 30–1000-times higher compared with other cell types. It was hypothesised that P-glycoprotein (p-gp) activity (multixenobiotic resistance, MXR) contributed to the resistance to OA. Vincristine and rhodamine B was used as p-gp substrates and verapamil or staurosporine (ST) as inhibitors of p-gp transport. However, no indications of cell membrane p-gp activity were detected. Instead, experimental observations led to the conclusion that a MXR transport system was present within lysosomal membranes. Various concentrations of OA did not affect the dynamics of vincristine in blood cells. As a positive control for the assay, p-gp activity was measured in mussel gill tissue. The efflux of rhodamine B was reduced by verapamil, which is, considered evidence for cell membrane p-gp activity, thus the accuracy of the method was confirmed. Rhodamine B efflux was also reduced by OA in gill tissue, which suggested that OA is either a competitive substrate or inhibitor of p-gp activity. When the volume of the lysosomal compartment was measured in blood cells pre-exposed to OA, a significant increase was detected compared with control cells. It was proposed that uptake and storage of OA within the lysosomal system might protect mussel blood cells from the cytotoxic effects of this compound.

Depuration of Okadaic acid (Diarrhetic Shellfish Toxin) in mussels, Mytilus edulis (Linnaeus), feeding on different quantities of nontoxic algae

Depuration of mussels contaminated by Diarrhetic Shellfish Toxins (DST) is a potential option for the shellfish industry to manage the impact of DST. Field observations have suggested that the main factor regulating the rate of depuration of DST is the quantity of nontoxic algae available for the mussels to feed upon. In this paper, the effects of the quantity of food, which mussels feed upon, on the rate of depuration of DST in Mytilus edulis L. was tested in a laboratory experiment. Mussels naturally contaminated by the DST okadaic acid (OA) were collected from a mussel farm located on the Swedish west coast during a bloom event. Individual mussels were placed in filtered seawater and given daily rations of a mixture of nontoxic algae as follows: no food, 0.5% and 1.5% of dry weight body mass day −1. Depuration was performed over 1, 2, 4, 8, 16 or 32 days. The levels of OA decreased in all treatments with time, with an average of approximately 50% reduction after 32 days. No significant differences in content of OA among food rations were detected. In contrast to predictions, a trend towards lower levels of toxins in the mussels receiving no food compared to both food treatments was observed after 32 days of depuration. The loss of toxins in mussels that were not feeding correlated with a considerable loss in the mass of the digestive gland between 16 and 32 days. It was concluded that the rate of depuration of OA in mussels is not positively correlated with digestive activity and fecal production. Instead, the lipophilic character of the OA molecule suggests that OA may have affinity for lipid-rich cellular and intracellular components. Increased usage of lipid stores, which occur during starvation, may accelerate the release of OA. This model could explain the observations made during the last part of this experiment. In management of toxic mussels, depuration in waters free of toxic algae is not likely to be enhanced by increasing the food supply to mussels; however, long periods of depuration in the absence of food should be avoided because of the negative effects on the condition of the mussels.

Comparing carcinogenicity and acute toxicity for ingestion of the promoting agent okadaic acid

Okadaic acid (OA) is a promoting agent present in mussels and other shellfish. While denatured by cooking, ingestion of raw mussels can lead to intakes of OA. To date, countries such as France have relied on measures of acute toxicity in regulating residual concentrations of OA in mussels. Recently, the French Government has moved to consider the carcinogenic risk from OA in mussels to ensure that regulations based on acute toxicity are not allowing concentrations of OA that might induce significant risks of cancer. This paper explores the carcinogenic risk posed by ingestion of OA, using a model of the biokinetics of OA following ingestion and a model of the promoting effects of OA. Within the carcinogenicity model, OA alters the balance between rates of cell mitosis and death, allowing selective expanasion of colonies of cells initiated by background events. Results of this preliminary, conservative, study indicate that current regulations based on acute toxicity probably are protective of public health, including protection against the risk of cancer.

Methodological improvement of the protein phosphatase inhibition assay for the detection of okadaic acid in mussels.

A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.

Semi-automated membrane based chemiluminescent immunosensor for flow injection analysis of okadaic acid in mussels

A chemiluminescent immunosensor integrated in a flow injection analysis system was developed for the detection of the ‘diarrheic shellfish poisoning’ (DSP) toxin okadaic acid (OA). Anti-OA monoclonal antibodies were labelled with horseradish peroxidase for their use in a competitive assay, in which the free antigen of the sample competes with immobilised OA. Based on commercially available polyethersulfone membranes, this bioanalytical system exhibits low non-specific binding of antibodies in the presence of mussel homogenate. The immunosensor was used in a semi-automated analysis procedure in which the free OA containing sample was injected in the flow system concomitantly with the labelled antibodies. With an overall measurement time of 20 min, the immunosensor has a detection limit of 0.2 μg OA/100 g mussel homogenate (0.1 μg OA/L in the assay solution), and measurements could be performed over three decades, from 0.2 to 200 μg OA/100 g of mussel homogenate. The operational stability of the sensor for the detection of critically contaminated mussels (40 μg/100 g of mussel homogenate) has been investigated over 38 OA determination cycles. A stable response was obtained under the 34 first measurements with a CV of 11.7%. Moreover, no significant change in the immunosensor performances was observed during short and long term storage periods of the membranes (1 month). The performances of five immunosensors (five different membranes) showed a good repeatability with a variation of the negative control and the critically contaminated signal equal to 7% and 12.6%, respectively.

Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate ( p-NPP) and the bioluminescence assay using luciferin phosphate (L–P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC 50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC 50 values of 0.9 and 6 nM using L–P and p-NPP respectively. CDP- star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC 50 for the colorimetric method (IC 50=2 nM [ p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations <1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels ≤1 μg/100 g of mussel tissue which is well below the limit of 20 μg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to 78% ( p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC–MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC–MS all scored false negative results compared to those for the mouse bioassay in the range 20–40 μg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay>ELISA>PP-2A>LC–MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 μg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC 50 being inversely related to the exposure time ( IC 50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC 50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.

Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhetic shellfish poisoning toxins in mussels

R. Draisci, L. Croci, L. Giannetti, L. Cozzi, L. Lucentini, D. De Medici and A. Stacchini. Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhoetic shellfish poisoning toxins in mussels. Toxicon 32, 1379–1384, 1994.—Mussel specimens ( Mytilus galloprovincialis) collected from two different areas of the Adriatic Sea were analysed for diarrhoetic shellfish poisoning (DSP) toxin by three methods: mouse bioassay, the DSP Check enzyme immunoassay kit, and high-performance liquid chromatography (HPLC). The results obtained confirm that Yasumoto's mouse bioassay, capable of detecting all the components of the DSP group, is still necessary to determine the wholesomeness of the product. The ELISA method has not always given quantitatively reliable results. The HPLC method is advantageous in terms of sensitivity, accuracy, specificity and rapidity. However, its application is limited so far to the determination of okadaic acid in mussels.

Okadaic acid in mussels of Adriatic sea

The diarrethic shellfish poisoning from mussels, collected in the main producing areas in the seawater off the coast of Emilia-Romagna (Italy), has been investigated. The ether soluble material from hepatopancreas of highly toxic mussels ( Mytilus galloprovincialis) was partitioned between n-hexane and methanol-water 9:1. The hydromethanolic phase was successively subjected to repeated chromatographic separations (SiO 2, Sephadex LH-20 and reversed-phase HPLC). Following the toxicity of each fraction by mouse lethality test, the presence of okadaic acid has been evidenced through 1H NMR spectroscopy. The investigation of two further toxic fractions is still in progress. The obtained results represent an initial contribution to current Italian legislation and to future health measures to be defined at European Community level.

Seasonal, geographic and individual variation of okadaic acid content in cultivated mussels in Sweden.

In Western Europe the dinoflagellate toxin, okadaic acid (OA) has been the main cause of diarrheic shellfish poisoning (DSP). Chemical determination of OA in mussels by homogenization of the hepatopancreas, extraction, purification, reaction with 9-anthryldiazomethane (ADAM), HPLC-separation, and fluorometric quantification has been used for weekly monitoring of mussel growing farms and to control harvested mussels. Within a week, substantial rises (from 0.41 to 5.4 micrograms OA/g hepatopancreas) as well as great reductions (from 7.2 to 1.8 micrograms/g hepatopancreas) were recorded. The rapid rise implies that weekly sampling is not sufficient to ensure that mussels are free from toxic levels of OA. The rapid decrease reveals that efficient toxin clearance mechanisms exist in the mussels. Substantial OA clearance occurs also at low temperatures (1.4-3 degrees C). Within a mussel growing site the OA concentrations could differ considerably between adjacent mussels (0.63 and 4.2 micrograms OA/g hepatop.) and even more between mussels grown at different depths along the same rope (0.63 and 10 micrograms OA/g hepatop.). These data emphasize the importance of sampling in studies on DST in mussels. Great differences between the different mussel growing sites were also observed. These data have been discussed with respect to the spread of the toxin by the sea, and the possibilities of reducing the exposure of the mussels to the toxic algae.